Vol 95, No 4 (2018)

Aim. To evaluate the metabolic activity of highly antagonistic strains of lactobacilli isolated from the oral cavity and intestine of healthy people. Materials and methods. 9 highly antagonistically active strains of lactobacilli isolated from plaque and intestine of healthy people of different age groups from 8 to 35 years were included in the study. Enzymes of pathogenicity, acidproduc-tion, gasotransmitters (CH₄, CO₂, C₂H₆, CO и NH₃) were studied in lactobacilli, as well as the degree of sensitivity to hydrochloric acid and bile. Results. All antagonistic strains of lactobacilli have been identified by 16S rRNA sequencing and assigned to 4 species: Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus casei. All lactobacilli were apatogenic, producing a wide range of metabolites of varying severity: lactic acid and gasotransmitters, CH₄, CO₂, C₂H₆, CO и NH₃. It was found that intestinal strains of lactobacilli were resistant to hydrochloric acid and bile than lactobacilli isolated from oral cavity. Conclusion. Highly antagonistic strains of lactobacilli have a wide spectrum of probiotic factors, they synthesize acids and gasotransmitters, persiste in the presence of hydrochloric acid and bile, and therefore they should be considered in the creation of new probiotics and functional products.

Aim. Evaluation of the effect of metabolites of vaginal lactobacilli on the enzymatic and bactericidal activity of muramidase. Materials and methods. We investigated how the enzymatic and bactericidal activity of muramidase changes after treatment with supernatants of lactobacilli containing surfactants, hydrogen peroxide, or a combination thereof. The enzymatic activity was measured by the rate of Micrococcus luteus lysis, bactericidal activity for test strains of Escherichia coli and Lactobacillus acidophilus by results of seeding on agar plates. Results. The hydrogen peroxide and surfactants reduced the enzymatic activity of lysozyme. Bactericidal activity of lysozyme against L. acidophilus and E. coli was decreased under the influence of surfactants. Hydrogen peroxide and its combinations with surfactants decreased bactericidal activity of lysozyme against L. acidophilus and increased for E. coli. Low concentrations of surfactants potentiated the effect of H₂O₂ on the antibacterial activity of lysozyme. Conclusion. It is concluded that the metabolites of the normal microflora is a implement for modification of host defense factors in order to create favorable conditions for its own existence, and prevent introduction of allochthonous species.

Aim. The construction of recombinant attenuated Bordetella pertussis bacteria of ptxP3 genotype and its genetic and biological stability characteristics. Materials and methods. During construction of recombinant attenuated bacteria of ptxP3 genotype virulent Bordetella pertussis bacteria of ptxP1 genotype were used as a recipient. PtxP1 genotype bacteria are used for whole cell pertussis (wP) vaccine production in Russia. Mutant bacteria B. pertussis 475 were received by crossing-over between chromosome comprising native copy of target sequence and its mutant copy in recombinant suicide plasmid transferred in recipient bacteria by conjugation. Genetically engineered construction of recombinant plasmids was conducted. The structure of modified chromosome locus of attenuated bacteria was determined by PCR and amplification fragments sequence. The stability of structure and characteristics of attenuated bacteria was defined after 15 passages of strains on culture medium and 5 passages in mice. Results. Isogenic attenuated ptxP1 B. pertussis 4M and ptxP3 B. pertussis 4MKS were constructed. These bacteria produce non-toxic pertussis toxin (PT) and do not produce dermonecrotic toxin (DNT). The promoter region of ptx operon contains mutation, typical for «new» genotype of circulating virulent bacteria and increasing PT production. The structure of modified DNA fragments and characteristics of attenuated bacteria did not change while storing and after passages on culture medium and in mice. Conclusion. Recombinant attenuated bacteria B. pertussis 4MKS of «new» ptxP3 genotype are constructed. Application perspectiveness of genetically engineered modification of isogenic B.pertussis bacteria for pertussis vaccines development is shown.

Aim. Of the study is to assess cervical mucus neutrophils functional status depend on dominant lactobacillus strain in I trimester of pregnancy women. Material and methods. We definded 40 lactobacillus strains obtained from the genital tract of the pregnant women. We used mass spectrometry analysis to identify vaginal lactobacilli and evaluated biofilm formation. Also, we studied cervical mucus neutrophils viability, lysosomal activity, spontaneous and induced NBT test (Nitroblue Tetrazolium test), ability to absorb the latex particles. Isolated strains were divided into 3 groups depending on presence or absence of vaginal or cervical channel infectious diseases. Results. We identified 5 lactobacilli types: L. jensenii, L. mspatus, L. аcidophilus, L. gasseri, L. delbrueckii among 40 definded strains. We determined different intensity of phagocytosis depending on dominant strain in normocenosis. With the L. crispatus prevalence demonstrated the least intensity of phagocytosis compared with L. acidophilus and L.jensenii. Lactobacilli able to intensively produce biomatrix in condition of genital infection. Thus blocking antigenic potential and result in absence of NBT-reducing power of neutrophilic granulocytes and reduction antimicrobial protection of pregnant women genital tract mucosa. Conclusion. The indicators of neutrophils functional status depend on dominant lactobacilli strain in I trimester of pregnancy women.

Aim. To assess the structure of the intestinal microbial consortium and the level of oxytocin in blood plasma in the context of severe social insufficiency in children with autism spectrum disorders. Materials and methods. 44 children with autism spectrum disorders were divided into two groups: 23 children with no signs of social contact and 21 people with preserved social contact. The comparison group was consisted of 39 typically developing children of the appropriate gender and age. The structure and quantity of microorganisms of the small intestine was determined with the help of specific lipid markers in peripheral blood by gas chromatography-mass spectrometry of microbial markers. The concentration of oxytocin was assessed in plasma by the method of ELISA. Results. Normally, the biochemical signals of the ecological system (the host-associated microbial consortium and the neuropeptide-oxytocin) work in the general context of the socialization of the meta-organism. However, an imbalance of the system (disease - autism spectrum disorders) can lead to change context and social insufficiency. Conclusion. Research in this direction will help, as we believe, to understand of the mechanisms underlying social deficits in autism, which will allow us to determine the ways of their correction.

Aim. To study the dependence of detection of markers of opportunistic infections from afherence to antiretroviral therapy in children born to HIV-infected mothers on the example of herpesvirus infectionsand pneumocystis. Materials and methods. Samples of biological materials (blood serum and blood cells) of 66 children with HIV infection aged 1 month to 15 years old were treated in Children’s Boxed Department of Children’s Hospital No. 2 with diagnoses «incomplete HIV test» (children from the age of one month to one and a half years) and «HIV infection». To determine IgM and IgG to herpesviruses and pneumocyst, the method of enzyme immunoassay was used; indirect immunofluorescence reaction for the detection of herpesviruses and their antigens in the blood, early antigens and virus reproduction were determined using a rapid culture method. Results. 56.0% of the surveyed children received complete antiretroviral therapy, in 16,7% of cases they were not complete, and 27,3% of children did not fully adhere to ARVT. Despite the fact that 100% of children with an incomplete diagnosis of HIV infection were covered by ARVT due to the use of chemotherapy drugs by their mothers during pregnancy, they still had markers of both active and latent forms of herpesvirus infections and pneumocystis. In children with confirmed HIV infection living both in social institutions and in families, the markers of opportunistic infections were more often diagnosed in patients receiving ARVT in full and not in full volume than in children who did not have it. Conclusion. Identification of markers of active forms of herpesvirus infections and pneumocystis in HIV-positive children not receiving ARV is the basis for its immediate appointment.

Aim. To indicate potentially pathogenic bacteria in plankton of the brackish rivers flowing into the Elton Lake by high-throughput sequencing of 16S ssuRNA gene. Materials and methods. The water samples from brackish rivers Lantsug and Chernavka, flowing into the Elton Lake, were taken up in a volume of 50 ml, filtered through membrane filters (pore diameter - 0.22 pm). Total DNAwas obtained by phenol-chloroform extraction with preliminary homogenization and enzymatic lysis. DNA libraries for sequencing were created by protocol Illumina with primers to a variable V3-V4 region of 16S ssuRNA gene. Sequencing was performed on a platform MiSeq («Illumina», США). Results.There were found the phylotypes of potentially pathogenic bacteria of Proteobacteria phylum from the families Enterobacteriaceae, Pseudomonadaceae, Campylobacteraceae, Vibrionaceae, Aeromonadaceae, Moraxellaceae, Legionellaceae, Alcaligenaceae, Campylobacteraceae, and also of Firmicutes, Bacteroidetes, Actinobacteria phyla in the plankton samples of the brackish rivers. Probable source of bacterial contamination is large and small cattle. Conclusion. These data demonstrate that the continental brackish waters, along with freshwater and marine habitats perform a reservoir function to potentially pathogenic microorganisms. High-throughput sequencing can be used to screen the presence of pathogens in water.

The in situ investigation of cyanobacterial seasonal succession, causing algal «blooms» in boreal waters, and its dependence on ecological factors was fulfilled. It was revealed that the most important abiotic factors promoting the growth and changes of dominant species in populations are the water temperature, the level of solar radiation and content of cyanobacterial metabolites. The most profound «blooming» with highest biomass and toxic metabolites accumulation occurs during anticyclone type of weather. During such periods cyanobacteria are the most prevalent cultures due to the combination of adaptive mechanisms acquired in evolutionary process which other partners of planktonic community lacked or deprived. It was hypotized that the global warming and cyanobacteria ability to inhabit different biotopes due to environmental tolerance would result in more profound and prolonged «blooming» of boreal waters.

Aim. Using the model of intragastric Listeria monocytogenes infection in pregnant mice to compare an input of found in nature variants of the invasion factor InlB in perinatal listeriosis. Materials and methods. Mice on 12-16 days of pregnancy were injected intragastrically with 10⁸ CFU of isogenic recombinant L. monocytogenes strains EGDeAinlB::InlB9 and EGDeAinlB:: InlB14. The strains expressed naturally occurring InlB variants, InlB9 and InlB14. In 72 h, mice were subjected to euthanasia to evaluate bacterial loads in the internal organs. Results. Only the strain, which expressed InlB14, caused perinatal infection. Microbial loads in the liver, spleen and Peyer’s patches was 715, 315 and 70 times higher for this strain than for the strain EGDeAinlB:: InlB9 (p<0,01). Microbial loads in the villous epithelium were comparable for both strains. Separate plating of placentas and fetuses infected with EGDeAinlB::InlB14 demonstrated that 5 and 2 placentas and fetuses, respectively, were infected from totally 9 embryonic structures. Conclusion. Naturally occurring InlB variants differed in their ability to provide perinatal listeriosis in intragastrically infected mice.